LowDose SPA

Goal:

Setup low dose modes.
View mode should be set up for automated hole centering.
Set camera parameters for all modes.
We want to collect as small as possible so uncorrected with frame alignment on the support pc and LZW zipped frames.

ALWAYS PUT THE SCREEN DOWN WHEN CHANGING OPTICS ON the K2, otherwise you will burn the camera!!!

We should still be in Nanoprobe mode.

SerialEM general settings

Buffer controls, Roll Buffers A -> H, nothing else selected:

Image Alignment & Focus control panel: check if "center image shift on tilt axis " is de-selected (we do not want unnecessary beam-image shift).
Image Alignment & Focus control panel: press ResetImageShift to reset any shift to zero.
.

Low Dose Control: switch on Low Dose Mode, select "continuous update of mag & beam".

Record mode setup

WITH THE SCREEN DOWN, Low Dose Control: "Area to show when screen down" select Record and adjust the beam and magnification with the Titan hand panels.
We mostly use Nanoprobe spot 6 or 7, aim for a camera dose rate of ~15 e/pixel/sec with the sample in the beam, the objective aperture inserted and the energy slit inserted.
If necessary, center beam in Titan UI with Direct Alignment/Tomo Beam shift.
Go to an empty area.
Take a Preview exposure.
SerialEM menu Calibration, Electron Dose:

Focus/Trial/Preview mode setup

Focus, Trial, Record and Preview will get the same optical settings:

Low Dose Control: while in Record mode, click on T and F in "Copy current area mag & beam to".
Make sure a 20 eV slit is inserted for all modes!

View mode setup

For View mode set a magnification that will cover just one entire hole, same spot size as used for Record/Focus/Trial?preview, -100 micron defocus.
If beam centering is off in View mode: in Low Dose Control, Additional Beam Shift, select Set, center beam with the left trackball, de-select Set. This stores an additional beam shift for just the View mode.
Low Dose Control: de-select "continuous update of mag & beam".

Focus/Trial distance and angle

Take a View image and position the Focus/Trial area conveniently on some carbon area by changing the Focus/Trial distance and the Rotate inter-area axis.

Camera settings for all modes

Camera & Macro Controls, Setup:

Low Dose mode	binning	size	Processing	K2 mode	exposure time	Dose fractionation
View	4	Full	Gain Normalized	Counting	0.1	No
Focus	4	Full	Gain Normalized	Counting	0.1	No
Trial	4	Full	Gain Normalized	Counting	0.1	No
Record	1	Full	Dark Subtracted	Counting	TBD	Yes, Save Frames!
Preview	4	Full	Gain Normalized	Counting	0.1	No

NOTE:
We align frames on-the-fly in IMOD with the GPU in the support PC, we save frames as small as possible, meaning Dark Subtracted and LZW-compressed tif.

Camera & Macro Controls, Setup, Record mode: check that Dose Fractionation Mode and Save Frames are selected only for Record mode, make sure you select:

Counting.
Bin counting frames by 2 in hardware.
Binning 1.
Processing, Dark Subtracted.
Dose Fractionation mode.
Save frames.

Set File Options:
TIFF (LZW compression), to keep the file size small.
Save unnormalised frames even if Gain nomalized is selected, to keep the file size small.
Save mdoc for each frame stack, contains all microscope and imaging conditions.
Save frames without rotation/flip to standard orientation, saves time.

Select a folder on your mapped group share, this is where you will run Warp later:

Check check!

Camera & Macro Controls: take a View, Trial, Focus and Preview image, twice in this order.

If the beam is off in Focus/Trial/Record/Preview: center it in any of these modes with Direct Alfgnment/Tomo Beam Shift.
If the beam is off in View: Low Dose Control, Additional Beam Shift, select Set, center beam with the left trackball, de-select Set.

Automated hole centering

SerialEM can do automated hole centering (thanks to Chen Xu at Brandeis). For this we store a Low Dose View image of a centered hole into an image buffer and align to this image when moving to a new target hole. For this we need to have Low Dose View and Record modes perfectly aligned.

SerialEM help file:

Shift offset for View.

If image features shift when going between the View and Record areas, the Set button allows you to correct for this shift. The Set button is enabled after you have shifted a View image into alignment with a Record or Preview image. The procedure for setting a shift is:

In the Image Alignment & Focus panel, turn off ‘Move stage for big mouse shifts’ if it is on.
Take a Preview image and use the right mouse button to center a feature that will be recognizable in the View image. Take another Preview image to verify that it is centered.
Take a View image. Use the right mouse button to center the feature.
Press Set in Low Dose Control. The shift will be recorded as a correction, and the program will take a new View image.
If the new View image is not centered, center the feature and press Set again. The new shift will be added to the existing one. Repeat if necessary.
Turn ‘Move stage for big mouse shifts’ back on.

If for some reason this shift offset becomes inappropriate, you can set it to zero with the Zero button.

Save a centered hole

If for whatever reason SerialEM needs to be restarted, you have to repeat this procedure!

Go to a nice grid square.
Run Eucentric Rough from Tasks menu.
Take a View image and center a typical hole with clean ice.
When it is centered you can save it in a file for later use, should SerialEM crash.
With the centered hole image as active image, click Copy Active image to Buffer P in control panel Buffer Controls:
The centered hole image is now stored in buffer P which can not be overwritten by acquisitions.

Open SerialEM menu Script, Edit/Run One Line..., type and run 'CallFunction HoleCentering':

Multi Shots

Take a View image.
Center a hole.
Open SerialEM menu Navigator, Montaging & Grids, Set Multi-shot Parameters, center the pattern in the middle of the centered hole in the View image:
Play with Number of shots and Distance from center, don't change other parameters.

For single shot per hole, simply deselect Do Multiple records: